Genome Analysis Of Water Spinach (Ipomoea Aquatica Forsk) And Genetic Effect Analysis After Genome Doubling

Water spinach（Ipomoea aquatica Forsk）belongs to Ipomoea genus of the Convolvulaceae.As a kind of high quality leafy vegetable,it is widely planted in China.The molecular research basis of water spinach was weak,the genome data was lacking,and the studies on gene function were few,which seriously affects the genetic improvement.In this study,we assembled the chromosomal-level nuclear genome of water spinach,obtained the complete chloroplast and mitochondrial genome,and performed comprehensive annotation and molecular evolution analysis.The autotetraploid water spinach was created by genome doubling technique,and comparative analysis of diploid and autotetraploid was carried out by using multidimensional omics technologies.The main results are as follows:1.Genome analysis of water spinachBased on the second-generation Illumina and third-generation Pacbio platforms,a total of 212.66 Gb sequencing data were obtained.The assembly results were as follows:The total length of contig is 511.50 Mbp,and contig N50 is 1.65 Mbp.The total length of scaffolds is 513.19 Mbp with the scaffold N50 of 2.75 Mbp.Using the GBS data and a F₂segregation population composed of 200 individual plants,these scaffolds were anchored to 15 pseudochromosomes with a anchoring rate of 81.6%.There were 56.20%repeats in the water spinach genome with the largest proportion（38.63%）.A total of 30,693protein-coding genes,1,316 mi RNA,1,832 t RNA,2,233 r RNA and 1,969 sn RNA were annotated.Phylogenetic analysis suggested that water spinach diverged from the common ancestor of three Ipomoea species（I.nil,I.triloba and I.trifida）about 7.1 million years ago.The expanded genes during the evolution of the water spinach were mainly enriched in Photosynthesis-antenna protein,oxidative phosphorylation,nucleotide resection repair,mismatch repair,homologous recombination and DNA replication pathways.The whole genome duplication（WGD）analysis indicated that the WGD event from water spinach occurred after divergence between Convolvulaceae and Solanaceae and before divergence between Ipomoea genus and Cuscuta genus.We also assembled and annotated the chloroplast genome of water spinach based on Illumina sequencing data from total DNA.The chloroplast genome showed a tetrad structure with a total length of 165,294 bp,GC content of 38.2%,containing 111 genes including 84 encoding protein genes,8 r RNAs and 19 t RNAs.The mitochondrial genome of water spinach was sequenced by second-generation Illumina and third-generation Prometh ION platforms with isolated mitochondrial DNA.The length of the mitochondrial genome was 296,746 bp,the CG content was 44.13%,and 64 genes were annotated,including 38 encoding protein genes,23 t RNAs and 3 r RNAs.In addition,8.49%of the mitochondrial DNA came from the chloroplast genome,including 9 complete genes.Collinearity analysis found that there were a few mutation sites between the sequenced mitochondrial genome and the published mitochondrial genome of water spinach.2.Creation of autotetraploid water spinach and comparative analysis of autotetraploid and diploid by multi-dimensional omics technologiesAutotetraploid water spinach was created by inducing water spinach cutting shoots with colchicine under open conditions.Compared with the diploid,in terms of external morphology,the leaves of autotetraploid water spinach were wider,the stems were thicker,and the roots were shorter and thicker.In terms of anatomy,there are bigger root tip meristem cells in autotetraploid water spinach,and xylem and phloem cells of autotetraploid water spinach increased in different degrees in the leaves and roots.The sponge cells,palisade cells of leaves from the autotetraploid also got bigger.The transmission electron microscope observation showed that autotetraploid water spinach had larger chloroplasts,and the scanning electron microscope observation showed that the stomata of autotetraploid water spinach were also larger;In terms of physiology and biochemistry,autotetraploid water spinach has stronger photosynthesis and antioxidation,and the contents of auxin IAA,nitrate nitrogen and vitamin C have also increased significantly.Comparative transcriptome analysis showed that differentially expressed genes were mainly enriched in starch and sucrose metabolism,amino sugars and nucleotide sugars metabolism pathways.Four genes involved in IAA synthesis（iaq.scaf＿876.254（IAA14）,iaq.scaf＿25.345（IAA16）,iaq.scaf＿73.877（AUX1）and iaq.scaf＿73.78）may play an important role.Ultraperformance liquid chromatography tandem mass spectrometry（UPLC-MS/MS）was used to analyze the metabolites in water spinach leaves,the number of Amino acids and derivatives was the largest,accounting for 16.82%of total metabolites.Differential metabolic analysis showed that the contents of phenolic acids,flavonoids,lignans,coumarins,terpenoids,quinones and tannins increased in the leaves of autotetraploid water spinach.Transcriptional and metabolic association analysis showed that differential metabolites and differentially expressed genes were associated with steroid biosynthesis,starch and sucrose metabolism,and linoleic acid metabolism.Whole Genome Bisulfite Sequencing（WGBS）technology was used to construct the DNA methylation map of autotetraploid and diploid water spinach,in which the methylation level of autotetraploid water spinach was higher than that of diploid water spinach,the average methylation level of TEs was much higher than that of genebody regions.A total of 12,281 Differentially methylated regions（DMRs）were identified between the autotetraploid and diploid water spinach,covering 3355 DMR-related genes,which were mainly involved in RNA degradation,plant hormone signal transduction and plant-pathogen interaction pathways.Chromatin immunoprecipitation technology and high-throughput sequencing technology were combined to campare the histone methylation of autotetraploid and diploid water spinach using H3K4me3 and H3K27me3 antibodies.It was confirmed that H3K4me3 modification mainly occurred at the transcription start site,while H3K27me3modification occurred in all genebody regions.KEGG enrichment results showed that differential peaks related genes were mainly enriched in plant hormone signal transduction,fatty acid metabolism,m RNA monitoring pathway and biosynthesis of secondary metabolites under H3K4me3 modification.Under H3K27me3 modification,differential peaks related genes were mainly enriched in m RNA monitoring pathway,plant hormone signal transduction,DNA replication,splicosome and biosynthesis of secondary metabolites.In summary,the genome of water spinach has been comprehensively analyzed by nuclear genome assembly and annotation,comparative genome analysis and organelle assembly and annotation Using the obtained autotetraploid water spinach,the genetic effect of doubling the water spinach genome was comprehensively explored.These results provide important data for molecular analysis of important characters of water spinach and comparative genome analysis among species,and provide important materials for breeding of water spinach.